1. Field of the Invention
The present invention relates generally to the field of tumor biology. In particular, the invention relates to a nucleic acid encoding a tumor suppressor and its use in inhibiting tumor growth. In one embodiment, the invention relates to expression constructs encoding p16 and their use in inhibiting cancer.
2. Description of the Related Art
Cancer is one of the leading causes of human disease, being responsible for 526,000 deaths in the United States each year (Boring et al., 1993). Lung cancer alone kills more than 140,000 people annually in the United States. Recently, age-adjusted mortality from lung cancer has surpassed that from breast cancer in women. Although implementation of smoking-reduction programs has decreased the prevalence of smoking, lung cancer mortality rates will remain high well into the twenty-first century. Unfortunately, current treatment methods for cancer, including radiation therapy, surgery and chemotherapy, are known to have limited effectiveness. The rational development of new therapies for lung cancer largely will depend on gaining an improved understanding of the biology of cancer at the molecular level.
With advances in molecular genetics and biology, it has become evident that altered expression of normal genes can lead to the initiation of transforming events that result in the creation of cancer cells. The conventional therapy for malignancy, such as chemotherapy and radiation, has focused on mass cell killing without specific targeting, often resulting in damaging side effects. A new direction in cancer therapy is to deliver a normal gene to replace or correct the mutated gene, thereby altering the malignant phenotype of transformed cells. Several expression constructs have been developed in order to deliver a gene into somatic cells with high efficiency.
Cells are regulated in both positive (stimulatory) and negative (suppressive) manners. Loss of negative regulation of cell growth is often found in malignant cells. Accumulating molecular genetic evidence has revealed that loss of negative regulators, or increase in positive regulators in normal cells, can produce such cellular growth abnormalities. Most negative regulators (Marx, 1993; Grunicke and Maly, 1993), referred to as tumor suppressors, have been found to be involved either in direct control of the cell cycle (e.g., Rb, p53, WT-1) or in the signaling pathway leading to cell growth and differentiation (e.g., NF-1). In addition, recent data suggest that genes related to the maintenance of cell architecture and polarity also may function as tumor suppressors (Marx, 1993; Fearon et al., 1990; Trofatter et al., 1993).
The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4 (CDK4), regulates progression through the G1. The activity of this enzyme may be to phosphorylate Rb at late G1. The activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the p16INK4 protein. p16INK4 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al., 1993; Serrano et al., 1995). Since the p16INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in hyperphosphorylation of the Rb protein. p16 also is known to regulate the function of CDK6.
p16INK4 belongs to a newly described class of CDK-inhibitory proteins that also includes p15B, p21WAF1, and p27KIP1. The p16INK4 gene maps to 9p21, a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the p16INK4 gene are frequent in human tumor cell lines. This evidence suggests that the p16INK4 gene is a tumor suppressor gene. This interpretation has been challenged, however, by the observation that the frequency of the p16INK4 gene alterations is much lower in primary uncultured tumors than in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994; Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994; Mori et al., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al., 1994; Arap et al., 1995). Restoration of wild-type p16INK4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).